Title

Rh-RhAG/ankyrin-R, a new interaction site between the membrane bilayer and the red cell skeleton, is impaired by Rh(null)-associated mutation.

Document Type

Article

Publication Date

2003

Keywords

Ankyrins, Blotting-Western, Calmodulin-Binding-Proteins, Cytoplasm, Detergents, Electrophoresis-Polyacrylamide-Gel, Erythrocytes, Flow-Cytometry, Glutathione-Transferase, K562-Cells, Lipid-Bilayers, Membrane-Glycoproteins, Mice-Inbred-BALB-C, Mice-Mutant-Strains, Models-Biological, Octoxynol, Protein-Binding, Protein-Structure-Tertiary, Recombinant-Fusion-Proteins, Rh-Hr-Blood-Group-System, Transfection, Two-Hybrid-System-Techniques

JAX Source

J Biol Chem 2003 Jul; 278(28):25526-33.

Abstract

Several studies suggest that the Rh complex represents a major interaction site between the membrane lipid bilayer and the red cell skeleton, but little is known about the molecular basis of this interaction. We report here that ankyrin-R is capable of interacting directly with the C-terminal cytoplasmic domain of Rh and RhAG polypeptides. We first show that the primary defect of ankyrin-R in normoblastosis (nb/nb) spherocytosis mutant mice is associated with a sharp reduction of RhAG and Rh polypeptides. Secondly, our flow cytometric analysis of the Triton X-100 extractability of recombinant fusion proteins expressed in erythroleukemic cell lines suggests that the C-terminal cytoplasmic domains of Rh and RhAG are sufficient to mediate interaction with the erythroid membrane skeleton. Using the yeast two-hybrid system, we demonstrate a direct interaction between the cytoplasmic tails of Rh and RhAG and the second repeat domain (D2) of ankyrin-R. This finding is supported by the demonstration that the substitution of Asp-399 in the cytoplasmic tail of RhAG, a mutation associated with the deficiency of the Rh complex in one Rhnull patient, totally impaired interaction with domain D2 of ankyrin-R. These results identify the Rh/RhAG-ankyrin complex as a new interaction site between the red cell membrane and the spectrin-based skeleton, the disruption of which might result in the stomato-spherocytosis typical of Rhnull red cells.